Isolation of a calcium-regulatory protein from black pigment gallstones: similarity with a protein from cholesterol gallstones.

We have previously isolated from 13 cholesterol gallstones a low molecular weight acidic bili-protein that inhibited the precipitation of calcium carbonate in vitro. We now report the isolation of a similar protein from seven black pigment gallstones. Cholesterol was removed from the stones by Soxhlet apparatus with methyl t-butyl ether, and bile acids were extracted with methanol. The protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after demineralization of the stones with ethylenediaminetetraacetate. Structural and functional properties of the protein from the black stones that were similar to the protein from the cholesterol stones included the following: (a) an apparent molecular weight of about 5 kD; (b) a high content of acidic (19.8%) and hydrophobic (50.1%) amino acids with a low content of basic residues (8.4%) and little sulfide-containing amino acids (1.9%); (c) an inhibitory effect on both the initiation and growth of calcium carbonate crystals in vitro; and (d) very tight (possibly covalent) binding of a diazo-positive yellow pigment, presumably bilirubin, with maximum spectral absorbance at 410 nm. The structural and functional similarities of these bili-proteins from black pigment and cholesterol gallstones and their striking effects on calcium carbonate precipitation in vitro suggest that they play a common role in the regulation of precipitation of calcium salts during the formation of both types of gallstones.